The overall objective of this work is to elucidate changes in differentiated function of normal human fibroblast-like cells occurring concomitantly with loss of proliferative capacity in vitro and to investigate possible mechanisms responsible for phenotypic alterations of aging cultures. Several parameters of collagen metabolism will be measured in mass populations of human diploid fibroblasts at various times during the process of in vitro aging, i.e., loss of proliferative potential, and in clonal populations derived from these mass populations in order to assess the extent of phenotypic variation in collagen metabolism among cells initiating the cultures and among cultures differing in age. Parameters to be investigated include: 1) rate of collagen synthesis, 2) rate of collagen degradation, 3) type of collagen produced and 4) effect of hydrocortisone on rate of synthesis, degradation, and type of collagen. Collagen production will be measured as collagenase sensitive 3H-pro and 3H-hypro incorporation following labeling with 3H-pro. Collagen degradation will be measured by determining nonprotein 3H-hypro. Collagen type will be assessed by separation of the procollagen gamma chains using polyacrylamide gel electrophoresis. The results of these experiments should indicate whether changes in collagen metabolism occur during the process of cellular senescence, and whether observed changes are due to selection of particular cell types or to linear differentiation.